摘要：

微流控液滴,具有体积小,单分散性好,通量高等特点,作为一种新型微反应器,被广泛应用于化学合成,蛋白质结晶,生物分子检测等研究领域,其中数字化检测是液滴的一个重要应用方向.液滴数字化检测主要以核酸为研究对象,通过将DNA/RNA以单分子的浓度分散入液滴中,进行生化反应实现信号放大,对反应后的液滴进行检测,由信号的强弱来判断液滴中目标物的有无,实现单分子水平的检测和浓度的绝对定量,为基因突变,SNP分析,痕量核酸检测等提供了一种有效的方法.数字化检测通常要求较高的通量(104~106),因此需要合适的高通量检测平台来实现对大量液滴的检测和分析.另一方面,在医学领域,肿瘤标志蛋白的检测已经成为一种重要的癌症早期诊断方法.一般肿瘤标志物蛋白在血液中含量极低,大约为10-16~10-12mol/L,目前对于蛋白质的单分子数字化检测仍然少有报道,发展一种针对痕量蛋白质的单分子水平检测方法对于癌症诊断具有十分重要的意义. 基于此,本论文搭建了液滴微流控荧光检测平台,实现对液滴荧光信号的高通量高灵敏检测,并与酶催化的信号放大方法相结合,把平台应用于单分子β-半乳糖苷酶的检测,将高通量数字化检测技术由核酸拓展到了蛋白质领域. 本论文主要包含以下研究内容: (1)搭建基于微流控芯片的液滴荧光检测平台.平台包括液滴生成和检测芯片,光学系统,信号采集及数据处理系统.平台使用473nm小型半导体激光器作为光源,改造的倒置荧光显微镜作为光路,在微流控芯片中对待检测的液滴逐一激发,使用光电倍增管检测其荧光信号,检测到的信号由NI数据采集卡进行采集,LabVIEW程序进行处理,最后使用Origin软件进行统计分析.通过对平台进行性能测试,其检测通量达到100droplets/sec以上,检测信号强度与荧光素浓度呈现良好的线性相关,对荧光素的检测限为10-10mol/L. (2)利用搭建好的液滴微流控荧光检测平台对β-半乳糖苷酶进行高通量的数字化检测.首先使用芯片生成包含单拷贝酶分子的液滴.酶分子催化非荧光底物(FDG)生成荧光素,使得液滴荧光信号增强,不含有酶分子的液滴则只具有底物微弱的背景荧光.检测反应后液滴的荧光信号,由信号的强弱来判断液滴中酶分子的有无,实现对单酶分子的检测,并通过泊松分布计算,对酶浓度进行绝对定量.分别对稀释成不同浓度的酶溶液进行检测,每种浓度检测10000个以上的液滴,酶浓度分析结果与稀释倍数呈现了良好的线性相关,R2=0.9997,表明了该平台用于单酶分子检测的准确性. 本论文研制了液滴微流控荧光检测平台,并实现了对β-半乳糖苷酶的高通量单分子检测.使用该方法发展高通量单分子ELISA,可以实现痕量疾病标志蛋白的检测.同时将各种信号放大方法与该平台相结合,进行高灵敏,高通量的单分子分析研究,在稀有突变基因检测,microRNA检测等现代生物分析领域具有广泛的应用前景.Microfluidic droplets, as a kind of new micro-reactors with small volume, monodispersity, and high-throughput characteristics, have been widely used in chemical synthesis of small molecules, protein crystallization, detection of biological macromolecules and other research areas. Digital detection is one of the most promising applications among them. Droplet-based microfluidic technique provides a novel method to detect and quantify DNA and RNA molecules in the biological samples. Single copy of DNA/RNA molecule was first encapsulated into monodisperse droplets in an oil-based emulsion. After finishing the biological reaction in the droplets, a single nucleic acid molecule can be detected and the absolute concentration of the sample can be quantified. This promising technology can be used for gene mutation detection, SNP analysis and trace nucleic acid detection. Digital detection usually require high-throughput (104~106), so an appropriate high-throughput detection platform to realize the detection and analysis of a large number of droplets is needed. On the other hand, the detection of protein-related tumor markers is a great challenge for early diagnosis of cancer. In general, the expression level of tumor marker protein in the blood is very low, at about 10-16~10-12 mol/L. The development of single-protein detection is very important for cancer diagnosis. Unfortunately, the detection technology for single protein has rarely been reported yet. In this paper, to achieve single protein detection, we first set up a microfluidic chip based droplet fluorescence detection platform. β-galactosidase is chosen as the model molecule for the single enzyme detection. By using our platform, high-throughput digital detection of single enzyme molecule is achieved. This thesis includes the following research projects: (1) Building a microfluidic-chip-based droplet fluorescence detection platform, including the chips for droplet generation and detection, optical system, signal acquisition and data processing system. We used a 473 nm semiconductor laser as the excitation source, and a converted inverted fluorescence microscope as the optical path. The droplets after reaction were injected into the detection chip, and single droplet fluorescence signal was collected by a photomultiplier tube. We used NI data acquisition card to acquire the signals, LabVIEW program to process the data, Origin software to analyze the results. By using our proposed platform, over 100 droplets can be detected per second, with good detection linearity of fluorescein. The detection limit was achieved as low as 10-10 mol/L. (2) Using the microfluidic droplet fluorescence detection platform for single β-galactosidase molecule detection. A single copy enzyme molecule, β-galactosidase, was encapsulated in the droplet, where the non-fluorescent substrate (FDG) can be catalyzed into fl

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